In the last 20 years, conjugate vaccines, comprising bacterial capsular polysaccharides conjugated to protein carriers have developed. Examples include the Haemophilus influenzae type b (Hib) conjugate vaccine [1] as well as conjugate vaccines against Streptococcus pneumoniae [2] and serogroup C Neisseria meningitidis (MenC) [3].
The carrier proteins used in licensed vaccines include tetanus toxoid (TT), diphtheria toxoid (DT), the nontoxic CRM197 mutant of diptheria toxin, and the outer membrane protein complex from group B N. meningitidis. Ideally, a carrier protein induces strong helper effect to a conjugated B-cell epitope (e.g. polysaccharide) without inducing an antibody response against itself. The use of universal epitopes, which are immunogenic in the context of most major histocompatability complex class II molecules, is one approach towards this goal [4]. Such epitopes have been identified within TT and other proteins. Alternatively, multi-epitope carrier proteins may be used, such as those described in reference 5.
Once a saccharide antigen has been conjugated to a carrier protein, the reaction mixture should be purified to remove free carrier protein that has no saccharide antigen conjugated thereto and unconjugated saccharide.
Various methods for the purification of free and conjugated carrier protein are known in the art, including hydrophobic chromatography, tangential ultrafiltration, diafiltration etc. [see also refs. 6 & 7, etc.]. However, these methods suffer from various drawbacks. For example, reference 8 proposes the use of gel filtration to purify GBS conjugates. However, this method requires a large volume of gel filtration matrix and is difficult to apply at a manufacturing scale. An alternative method that involves ultrafiltration, for example using tangential flow diafiltration with a 100 KDa membrane, is not effective unless the saccharide antigen is of a suitably high molecular weight and is thus not suitable for GBS serotype III conjugates or other conjugates where the saccharide antigen is <100 kDa. In addition, ultrafiltration can result in a low yield and stress the conjugate.
It is therefore the object of the invention to provide an improved method of purifying saccharide antigen-carrier protein conjugates from impurities such as unconjugated carrier protein and unconjugated saccharide.